The study reports the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspsase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively, and this process promotes APAF-1 oligomerization into a large multimeric complex. This complex, isolated by gel filtration chromatography, is sufficient to recruit and activate procaspsase-9, with a stoichiometric ratio of procaspsase-9 to APAF-1 of approximately 1:1. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3. The study also demonstrates that dATP/ATP binding and hydrolysis by APAF-1, as well as cytochrome c binding, are crucial for procaspase-9 activation. The formation of this APAF-1-cytochrome c complex is proposed to be a functional apoptosome, playing a key role in the activation of caspases and the characteristic features of apoptosis.The study reports the reconstitution of the de novo procaspase-9 activation pathway using highly purified cytochrome c, recombinant APAF-1, and recombinant procaspsase-9. APAF-1 binds and hydrolyzes ATP or dATP to ADP or dADP, respectively, and this process promotes APAF-1 oligomerization into a large multimeric complex. This complex, isolated by gel filtration chromatography, is sufficient to recruit and activate procaspsase-9, with a stoichiometric ratio of procaspsase-9 to APAF-1 of approximately 1:1. Once activated, caspase-9 disassociates from the complex and becomes available to cleave and activate downstream caspases such as caspase-3. The study also demonstrates that dATP/ATP binding and hydrolysis by APAF-1, as well as cytochrome c binding, are crucial for procaspase-9 activation. The formation of this APAF-1-cytochrome c complex is proposed to be a functional apoptosome, playing a key role in the activation of caspases and the characteristic features of apoptosis.